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Diversity Generating Retroelements – Understanding a New Class of Mobile RNAs

Final Report Summary - RETROELEMENTS (Diversity Generating Retroelements – Understanding a New Class of Mobile RNAs)

Our research interest lies in studying retroelements, a ubiquitous class of genetic elements found in all domains of life. They have a major role in the evolution, organization and expansion of genomes. Retroelements are genetic elements that use reverse transcriptase to insert DNA copies of their RNA into new locations in the host genome. They comprise retrotransposons (mobile elements that replicate via an RNA intermediate, thus leading to the massive expansion and rearrangement of many genomes), group II introns (the evolutionary precursors of spliceosomal introns) and a number of other elements like retrons, mitochondrial plasmids, and a newly discovered class of viral and bacterial retroelements, the diversity-generating retroelements (DGRs).

DGRs have the unusual ability to repeatedly target and mutagenize a defined region of the host genome. A region of the genome (the template repeat TR) is transcribed into RNA and then reverse transcribed into cDNA: During this process, the reverse transcriptase introduces frequent errors, which lead to a new, mutated version of this sequence. This mutated cDNA is then used to replace the variable region (VR). Since the VR is usually part of the coding sequence of a gene, this leads to the expression of a protein with a modified amino acid sequence. Iterative repetitions allow the target protein to continuously change its properties. Such hypervariability can be exceedingly useful for the host organism because it allows fast adaptation to changing environmental conditions. Therefore DGRs are the first retroelements known to convey an immediate benefit to their host. Moreover, this mechanism also has great potential for biotechnology and medicine.

Previous research on DGRs had focussed almost exclusively on a phage DGR, and the distribution of DGRs in other realms of life was not clear yet. Thus we first developed an algorithm to automatically identify DGRs in the genomes of other organisms. By conducting an extensive database search, we could identify a total of 155 DGRs, 126 of which had not been described before (Schillinger, T. et al., 2012, Schillinger, T. and Zingler, N., 2012). Only a few of those were phage elements, while the overwhelming majority was found in prokaryotic genomes. In order to better understand the general rules underlying the mechanism of DGR activity, this project then focussed on developing in vitro and in vivo models of bacterial DGRs. We found that a certain cyanobacterial strain is particularly suited as DGR model organism: We could show that its DGR is currently actively mutagenizing the VR, and we were able to isolate, purify and biochemically characterize several of the DGR’s components This project therefore resulted in several significant advances towards understanding the basic principles of this novel mechanism driving evolution. The results will be disseminated in at least three more publications and lay the groundwork for examining further details of DGR activity. This field of research has many potential applications in medicine (immunology, phage therapy, DNA repair) and biotechnology (in vitro evolution, targeted DNA integration, developing reverse transcriptases with unusual properties, etc.).